The polysaccharide xanthan gum is produced by the microorganism Xanthomonas campestris and has the primary structure shown in Formula I, which is shown in FIG. 2.
Xanthan gum consists of a cellulosic backbone of β-1,4 linked D glucose units substituted on alternate glucose residues with a trisaccharide side chain. The trisaccharide side chain is composed of two mannose units separated by a glucuronic acid. Approximately half the terminal mannose units are linked to a pyruvate group and the non-terminal residue usually carries an acetyl group. The carboxyl groups on the side chains render the gum molecules anionic. Xanthan gum has a molecular weight of about 2×106 Daltons with a narrow molecular weight distribution compared to most polysaccharides. X-ray diffraction studies on xanthan gum fibres have identified a right handed, five fold helix conformation. In this conformation the side chains are aligned with the backbone and stabilise the overall conformation. In solution the side chains wrap around the cellulose-like backbone thereby protecting it. It is believed that this is responsible for the excellent stability of xanthan gum under adverse conditions.
Two of the key functionalities of xanthan gum that set it apart from other hydrocolloid thickeners are its rheology and its ability to interact synergistically with galactomannans.
The rheology can be characterised by the development of very high viscosity at low shear rates and pseudoplastic flow which provide suspension stability to finished products and low viscosity at higher shear rates for ease of filling, pouring and pumping.
Xanthan gum also has the ability to interact with galactomannans such as guar gum, cassia gum, tara gum and locust bean gum and with structurally similar polysaccharides such as the glucomannan konjac. This interaction results in either a synergistic increase in viscosity in the case of guar gum or the formation of strong self supporting gels as seen with locust bean gum and konjac glucomannan.
These functionalities are used in a wide range of food applications and maximising these through control of the xanthan structure and manufacturing process would provide a high performance xanthan gum of real benefit to the customer, in terms of cost in use.
Synergy
Initial studies (Morris, E. R., Rees, D. A., Young, G., Walkinshaw, M. D. and Darke, A. “Order-Disorder transition for a bacterial polysaccharide in solution. A role for polysaccharide conformation in recognition between Xanthomonas pathogen and its plant host.” J. Mol. Biol., 110 (1977) 1-16.) proposed a model in which the unsubstituted, galactose free (smooth) regions of the galactomannans (glucose free in the case of konjac mannan) bind to xanthan in its ordered state. This model has been used to explain the difference in degree of interaction between galactomannans of differing degrees of substitution. Subsequent studies, which showed that interactions were enhanced after heating the mixture to temperatures above the coil-helix transition of the xanthan, were interpreted as evidence that the binding occurred with the cellulosic backbone of the xanthan gum in the ordered state. An alternative explanation was that when the hydrocolloids are mixed at temperatures below the setting point of the gel, they form an inferior, disrupted network with melting and resetting giving a stronger, coherent gel.
The degree/strength of associations is increased as galactose substitution is decreased. For example locust bean gum which has a galactose content of 17-26% forms self supporting gels with xanthan gum whereas guar gum which has a galactose content of 33-40% forms weak gel networks resulting in a synergistic increase in viscosity. The molecular weight of the galactomannans is also known to influence their interactions with xanthan gum. The lower the molecular weight, the weaker the interactions seen with xanthan gum. It has been reported that increasing the ionic strength of the solvent reduces the associations between xanthan and galactomannans/glucomannans: for example increasing salt concentrations or lowering of pH results in weaker gels or lower viscosity in the mixtures.
Xanthan Molecular Structure and its Influence on Functionality
The role of the acetate and pyruvate groups in the molecular structure of xanthan gum and their impact on functionality is probably the most widely studied aspect of the structure-function relationship. Particular emphasis has been given to their role in controlling the rheology of the xanthan gum and their influence on interactions with the galactomannans. Assuming only one acyl group per side chain, the stoechiometric amounts of acetyl and pyruvate are 5.0% and 8.1% respectively. Xanthan gum sold commercially contains between 3 and 4% acetate and between 4 and 5% pyruvate. The strength of gels of xanthan and locust bean gum or konjac mannan have been shown to be very dependent on the degree of acetyl substitution (Wang, F., Wang Y.-J. and Sun, “Z. Conformational role of xanthan in its interaction with locust bean gum.” J. Food Science, 67 (2002) 2609-2614; Shatwell, K. P., Sutherland, I. W., Ross-Murphy, S. B. and Dea, I. C. M. “Influence of the acetyl substituent on the interaction of xanthan with plant polysaccharides—I. Xanthan—locust bean gum systems.” Carbohydr. Polym., 14 (1991) 29-51; Shatwell, K. P., Sutherland, I. W., Ross-Murphy, S. B. and Dea, I. C. M. “Influence of the acetyl substituent on the interaction of xanthan with plant polysaccharides—III. Xanthan—konjac mannan systems.” Carbohydr. Polym., 14 (1991) 131-147). Shatwell et al concluded that the interactions increase with decreasing acetylation. This resulted in stronger gels with LBG and konjac mannan, a result also seen by others. They also suggested that low-acetate xanthan had stronger interactions with guar gum compared to standard xanthan, something which has also been reported in several other studies. Morrison et al. showed evidence for this in the form of increased viscosity at 1 s−1 in low-acetate xanthan/guar mixtures compared to standard xanthan/guar mixtures.
There is very little evidence to suggest that the pyruvate content of xanthan has an influence on interactions with galactomannans. Shatwell et al indicated that gel strength with LBG reduced slightly with reduction of pyruvate level but results were not conclusive since the molecular weight of the low-pyruvate samples was lower than standard xanthan.
It has been shown by several workers that the pyruvate content of xanthan has a strong influence on the viscosity of the product. The viscosity increases with increasing pyruvate content. Flores and Deckwer suggested that there is not a continuous relationship between pyruvate content and viscosity, but rather, that there is a step increase when going from below 2% to above 3% pyruvate (Flores Candia, J.-L. and Deckwer, W.-H. “Effect of the nitrogen source on pyruvate content and rheological properties of xanthan.” Biotechnol. Prog., 15 (1999) 446-452).
It has also been demonstrated that the viscosity of low pyruvate xanthan is less sensitive to the addition of salts (Cheetham, N. W. H. and Norma, N. M. N. “The effect of pyruvate on viscosity properties of xanthan.” Carbohydr. Polym., 10 (1989) 55-60).
There is one published paper that contradicts this and claims that pyruvate content has no significant effect on solution viscosity and they attributed the differences observed by other workers to possible differences in molecular weight (Bradshaw, I. J. Nisbet, B. A., Kerr, M. H. and Sutherland, I. W. “Modified xanthan—its preparation and viscosity.” Carbohydr. Polym., 3 (1983) 23-38). However, in this study, viscosity was measured at shear rates between 8.8 and 88.8 s−1. These relatively high shear rates may account for the lack of difference in measured viscosity. Generally the viscosity differences are far more marked at shear rates below 0.1 s−1. Christensen et al (Christensen, B. E., Smidsrod, O and Stoke, O. “Xanthans with partially hydrolysed side chains: Conformation and transitions.” In Carbohydrates and Carbohydrate Polymers. Analysis, Biomedical and other Applications. M. Yalpini (ed.), ATL press (1993) pp 166-173) have shown that the terminal β-mannose is relatively susceptible to acid hydrolysis so low pyruvate samples prepared in this way may also have reduced molecular weight due to removal of this sugar. Since acid hydrolysis has been used to prepare low pyruvate xanthan samples in many of the referenced studies, further work is needed to separate the effects of molecular weight from the effects of pyruvate content.
The presence of acetate on the other hand tends to reduce xanthan gum viscosity (Hassler, R. A. and Doherty, D. H. “Genetic engineering of polysaccharide structure: Production of variants of xanthan gum in Xanthomonas campestris”. Biotechnol. Prog., 6 (1990) 182-187). It has been shown that an acetate-free xanthan has higher viscosity than native xanthan. Some work has been done on trying to identify distribution of acetate and pyruvate by fractional precipitation with ethanol to differentiate xanthan preparations into fragments with differing pyruvate content (Shatwell, K. P., Sutherland, I. W., Ross-Murphy, S. B. and Dea, I. C. M. “Influence of the acetyl substituent on the interaction of xanthan with plant polysaccharides—III. Xanthan—konjac mannan systems.” Carbohydr. Polym., 14 (1991) 131-147). It was found that as the alcohol level increased the pyruvate level in the precipitated fraction increased. This has also been achieved using an affinity matrix prepared by coupling antibodies to a Rizobium polysaccharide to a Sepharose gel column (Shatwell, K. P., Sutherland, I. W., Ross-Murphy, S. B. and Dea, I. C. M. “Influence of the acetyl substituent on the interaction of xanthan with plant polysaccharides—II. Xanthan—guar gum systems.” Carbohydr. Polym., 14 (1991) 115-130). Using this technique it was possible to identify a pyruvate-rich and pyruvate-poor fraction from the same xanthan preparation indicating some heterogeneity in the distribution of pyruvate. The implications of this for functionality were not discussed.
Methods to Control Xanthan Molecular Structure
A considerable amount of work has also been done on methods of controlling the levels of acetate and pyruvate during production of xanthan gum and Tables 1 and 2 summarise the techniques for the control of these substituent groups. These include the selection or modification of the Xanthomonas strain, control of certain parameters during the fermentation or post fermentation treatment during recovery of the gum.
TABLE 1A summary of the known techniques available forthe control of the acetate level in xanthanMethod of controlAdvantagesDisadvantagesPost fermentationHas been success-Variable acetate contenttreatmentfully performedReduction in MwHeat treatment atat pilot scaleSpecific enzymes notalkaline pH Enzy-describedmatic methodsGenetic control ofOK for 0% acetateNot suitable for inter-strain (KELTROL/Preservation of Mwmediate acetate levelsKELZAN ASX)GMO issue
TABLE 2A summary of the known techniques available forthe control of the pyruvate level in xanthan.Method of controlAdvantagesDisadvantagesFermentationConsistent pyruvateYield?conditionscontent (high or low)Nitrogen contentLow viscosity brothduring fermentationPreservation of MwOxygen availabilityPost fermentationNo GMO issuesVariable pyruvate contenttreatment HeatReduction in Mw (loss oftreatment at acid pHterminal β-mannose?) Spe-Enzymatic methodscific enzymes not describedGenetic control ofOK for 0% pyruvateNot suitable for inter-strainLow viscosity brothmediate pyruvate levelsPreservation of MwGMO issueStrains of Xanthomonas campestris 
Hassler and Doherty (cf. above) evaluated the properties of xanthan prepared from Xanthomonas campestris mutants obtained by genetic engineering. These strains carried a chromosomal deletion mutation that eliminated the entire gum gene cluster in their genome. The gum gene cluster was instead present in each strain on a recombinant plasmid. The gum gene mutations were present in these cloned gum genes. These mutants were defective in the xanthan biosynthetic pathway and produced xanthan that varied in the content and position of the acetate and pyruvate groups. The 6 possible variations are summarised in FIG. 1. They found that, when pyruvylation of the outer mannose is blocked by mutations that inactivate ketalase, high-level acetylation of the outer mannose results (variant 4). They concluded that variant 3 (high-pyruvate, low-acetate) gave the highest viscosity and that the presence of acetate decreased viscosity regardless of its position.
Viscosity measurements were made on xanthan recovered from broths that were not subjected to a thermal treatment. Therefore the viscosity values reflect those of the native polymer. No studies were made on other aspects of functionality such as salt tolerance, acid stability, hydration or interaction with galactomannans.
European patent application EP 0 765 939 discloses zero and low-pyruvate structures (variants 1 and 2), the strain that produces them and the process for production of the polymer.
U.S. Pat. No. 6,316,614 discloses all 6 variants, the strains that produce them and the process for production of the polymers.
Fermentation Process
The degree of pyruvylation appears to be particularly sensitive to the fermentation conditions and media. In particular the nitrogen source and oxygen availability have been shown to have an effect on pyruvate content. Peters et al. showed that the degree of pyruvylation decreased when the microbial oxygen demand was not met (Peters, H.-U., Suh, I.-S., Schumpe, A. and Deckwer, W.-H. “The pyruvate content of xanthan polysaccharide produced under oxygen limitation.” Biotechnology Letters, 15 (1993) 565-566). Flores Candia and Deckwer (cf. above) demonstrated that the pyruvate level was dependent on the level of NH4Cl in the fermentation media. They found that pyruvate content increased with decreasing nitrogen content in the media.
For example, at 8.4 g/l NH4CI, the pyruvate content remained constant at approximately 1.5% throughout the 140 h fermentation but at lower nitrogen levels (0.62 g/l) the pyruvate content came close to the theoretical maximum. The preparation of a high pyruvate xanthan gum through the control of the nitrogen content in the fermentation media is the subject of U.S. Pat. No. 4,394,447. The patent defines high pyruvate as at least 3.3% pyruvic acid by weight measured by an enzymatic method. This patent only discusses the fermentation conditions and no disclosure relates to the functionality or application of a high pyruvate xanthan gum.
Post Fermentation Treatment
Heat treatment of the fermentation broth at acidic pH's prior to recovery favours the removal of pyruvate groups. Heat treatment of the fermentation broth at alkaline pH prior to recovery favours the removal of acetate groups. Acetate can also be removed by treatment with alkali under nitrogen at room temperature.
Commercial Potential of Modified Xanthan
Commercially available xanthan products in which the acetate or pyruvate content has been deliberately controlled are available. CPKelco produces acetate-free products for food and non-food use called KELTROL ASX and KELZAN ASX, respectively. The main functionality benefit for this type of products is seen primarily in very strong acidic systems such as toilet cleaners where up to 5 to 10% strong acids are used giving the products a pH of around pH 2 or below. Significantly better long term stability is seen with the low acetate xanthan compared to a conventional product. In less acidic food products such as food dressings, the differences are much less significant. This product has greater synergy with galactomannans compared to standard xanthan and higher low shear viscosity than standard products. The product is made using a Xanthomonas strain that does not add the acetate group during fermentation (U.S. Pat. No. 6,316,614 B1).
To conclude, there is still a need to provide improved xanthan gums, in particular xanthan gums with improved rheological and/or synergy properties.